The major objective of this competitive renewal application is to elucidate the physiological significance of the sole member of the ankyrin (A) TRP subfamily, TRPA1, in endothelium-dependent regulation of cerebral arteries. Preliminary findings indicate that TRPA1 is present in the endothelium of human and mouse cerebral arteries, but is not present in other vascular beds, suggesting that TRPA1 plays a critical but unknown role in the regulation of the cerebral vasculature. Therefore, the overall goal of the proposed research is to determine the channel's function in healthy and diseased cerebral arteries. The goal of Specific Aim 1 is to identify endogenous activators of TRPA1 in the endothelium. This Aim will test the hypothesis that lipid peroxidation products (LPP) generated by NADPH oxidase (NOX) activity stimulate TRPA1 in the endothelium in an autocrine manner. We created the first endothelial cell-specific TRPA1 knockout (eTRPA1-/-) mice as a powerful experimental tool for these studies. Patch-clamp electrophysiology and an innovative total internal reflection fluorescent microscopy (TIRFM) method that we developed will be used to record the effects of endogenously-produced LPP on TRPA1 activity in native endothelial cells isolated from eTRPA1-/- and control mice. The goal of Specific Aim 2 is to elucidate the signaling pathways responsible for vasodilation evoked by endogenous TRPA1 activators. This Aim will test the hypothesis that LPP generation localized to myoendothelial junctions (MEJs) of cerebral arteries causes dilation by activating TRPA1 to initiate endothelium-dependent hyperpolarization (EDH). This hypothesis is supported by preliminary data showing that TRPA1 channels, NOX, and LPP are concentrated in MEJs, and that endogenously-produced LPP dilate cerebral arteries from control, but not eTRPA1-/- mice. Experiments for this Aim will use pressure myography and intracellular microelectrode recordings to examine the effects of exogenous and endogenously-generated LPP on arteries from control and eTRPA1-/- mice. The detailed structure of MEJs in cerebral arteries will be elucidated by immunolabeling experiments and proximity ligations assay. Subcellular Ca2+ signaling associated with TRPA1 activity will be recorded from arteries isolated from mice expressing the genetically-encoded Ca2+ indicator protein GCaMP2 selectively in the endothelium. The goal of Specific Aim 3 is to test the hypothesis that TRPA1-mediated EDH of cerebral arteries opposes elevated smooth muscle cell contractility associated with hypertension. We will test the hypothesis that basal TRPA1 activity is elevated in arteries from hypertensive animals vs. controls. Further, we will test the hypothesis that eTRPA1-/- mice will suffer stroke at higher frequency during hypertension compared with controls. These experiments will utilize the established renin-angiotensinogen (R+A+) transgenic model of hypertension. Proposed studies are expected to have substantial impact because they will elucidate the physiological function of TRPA1 in the endothelium of cerebral arteries and determine if TRPA1 is a potential therapeutic target for cerebrovascular diseases.